The long-term objective of this application is to determine mechanisms leading to excitation, activation and sperm entry in the egg at fertilization. The aims of research proposed here are to establish ultrastructural relationships between the interacting sperm and egg and correlate these observations with the electrophysiological responses that occur during excitation and activation. Experiments will be conducted to answer the following questions: (1) What is the ultrastructural relationship of sperm and egg components (e.g., sperm and egg plasma membranes, acrosomal process, bindin, vitelline layer, etc.) at the site of gamete interaction; (2) What is the relationship between the spatial organization of gamete components and electrical changes that occur during excitation and activation; and (3) Does sperm-egg fusion precede activation or possibly even excitation? Specific experiments will include electron microscopic observations carried out on inseminated eggs in which events of gamete interaction will be timed relative to the initiation of excitation (l(on)) in eggs clamped at different voltages or, treated with different agents known to affect sperm entry, excitation and/or activation. Investigations will be conducted to determine the ultrastructural relationships of sperm and egg components, especially their plasma membranes in preparations patched clamped for capacitance measurements. To gain further insights into changes that occur at the site where the gametes interact and fuse, this region will be examined with cyto- and immuno-chemical methods to demonstrate changes in specific sperm and egg components (e.g., plasma membrane, bindin and vitelline layer) and the passage of intracellular tracer from preloaded eggs into interacting sperm. This proposal will attempt to establish the primary events in these processes and determine morphological and electrophysiological relationships during gamete interactions leading to and following sperm-egg fusion. Because the research to be conducted is concerned with the fusion of the gamete membranes and the role of membrane potential in this process, new insights could be provided into membrane fusion processes including cell-cell fusion, exocytosis, the fusion of viruses with their host cells (an important area of consideration in AIDS research). The proposed research could provide information basic to the design of new contraceptive agents and an understanding of certain types of infertility.